NK (Natural Killer) cell and application of NK cell in tumor treatment medicine

Abstract

The invention relates to the technical field of biological medicine, in particular to an NK cell and application of the NK cell in tumor treatment medicines.The NK cell is prepared through the following method that a, an anti-PD-L1 antibody with the concentration being 20-35 micrograms / mL is used for pretreating a human peripheral blood mononuclear cell; b, in a container coated with the anti-CD16 antibody, a culture medium containing Galunissertib is used for culture, and the concentration of the Galunissertib is 4-6 [mu] M; and step c, transforming and expressing an F158V mutant CD16a receptor of UniProt P08637 through gene engineering. According to the technical scheme, a systematic solution is provided for NK cell therapy of a targeted tumor microenvironment, and wider clinical application in treatment of solid tumors and hematological tumors is expected to be achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to NK cells and their application in tumor treatment drugs. Background Art

[0002] The field of tumor immunotherapy has made significant progress in recent years, particularly in the development of drugs targeting immune checkpoint pathways such as PD-1 / PD-L1. Tumor cells often upregulate PD-L1 expression to bind to PD-1 on the surface of immune cells, thereby inhibiting the activity of T cells and NK cells and forming an immune escape mechanism. Anti-PD-L1 antibodies have been widely studied to block this signaling pathway and restore the ability of immune cells to attack tumors. However, strategies solely relying on PD-L1 blockade have limited efficacy in some patients, and other inhibitory factors in the tumor microenvironment may further impair immune cell function. Existing in vitro NK cell culture systems are often limited by issues such as expansion efficiency, survival rate, and functional maintenance. Targeted intervention against the multiple inhibitory factors in the tumor microenvironment requires further optimization. Summary of the Invention

[0003] In order to solve the problems in the related art, the embodiments of the present disclosure provide a NK cell and its application in tumor treatment drugs.

[0004] In a first aspect, the present disclosure provides an NK cell, which is prepared by the following method: Step a: Pre-treat human peripheral blood mononuclear cells with anti-PD-L1 antibody at a concentration of 20–35 μg / mL; Step b: Incubate cells in an anti-CD16 antibody-coated container with medium containing galunisertib at a concentration of 3–6 μM. Step c: Genetically engineer the CD16a receptor expressing the F158V mutant of UniProt P08637.

[0005] According to an embodiment of the present disclosure, the anti-PD-L1 antibody in step a is a humanized monoclonal antibody.

[0006] According to an embodiment of the present disclosure, the concentration of Galunisertib in step b is 4-6 μM.

[0007] According to an embodiment of the present disclosure, the genetic engineering modification in step c is achieved by lentiviral vector transduction.

[0008] According to an embodiment of the present disclosure, the culture medium in step b contains IL-2 at a concentration of 20-80 μg / mL.

[0009] According to an embodiment of the present disclosure, the anti-PD-1 antibody is a PD-1 / PD-L1 pathway specific blocking antibody.

[0010] According to an embodiment of the present disclosure, the culture medium in step b contains anti-CD2 monoclonal antibodies at a concentration of 100-500 ng / mL and anti-NKp46 monoclonal antibodies at a concentration of 100-500 ng / mL.

[0011] According to an embodiment of the present disclosure, the culture period is 13-15 days.

[0012] In a second aspect, the presently disclosed embodiments provide a use of NK cells as described in any one of the first aspects in the preparation of drugs for treating tumors.

[0013] According to an embodiment of the present disclosure, the drug includes NK cells and anti-PD-1 antibodies.

[0014] The technical effects provided by the embodiments of the present disclosure may include the following beneficial effects: According to the technical solution provided in the embodiments of the present disclosure, NK cells are prepared by the following method: Step a: pre-treating human peripheral blood mononuclear cells with an anti-PD-L1 antibody at a concentration of 20–35 μg / mL; Step b: culturing the cells in a medium containing galunisertib at a concentration of 4–6 μM in a container coated with an anti-CD16 antibody; Step c: genetically engineering the cells to express the F158V mutant CD16a receptor of UniProt P08637. In this technical solution, pre-treatment with 20–35 μg / mL of anti-PD-L1 antibody significantly blocks the inhibitory effect of PD-L1 on NK cells in the tumor microenvironment. Furthermore, galunisertib's TGF-β signaling blockade further relieves TGF-β-mediated immunosuppression, thereby enhancing NK cell activation and cytotoxicity. The combination of the anti-CD16 antibody-coated container and galunisertib not only promotes the directed expansion of NK cells but also maintains high cell purity while avoiding the reliance on serum components in traditional culture systems. The genetically engineered F158V mutant CD16a receptor significantly increases the binding affinity of NK cells to antibody drugs (such as anti-PD-1 antibodies), enhancing ADCC by over 30%, reducing receptor shedding, and prolonging the duration of the effect. This provides a systematic solution for NK cell therapy targeting the tumor microenvironment and is expected to achieve broader clinical application in the treatment of solid tumors and hematologic malignancies.

[0015] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure. DETAILED DESCRIPTION

[0016] The present invention is further illustrated below by way of examples, but it is to be understood that these specific examples are not intended to limit the scope of the present invention in any way. It should be noted that, unless otherwise specified, the raw materials used in the following examples are all commercially available.

[0017] Example 1

[0018] This embodiment provides a NK cell, which is achieved by the following steps: Pretreatment step: 50 mL of peripheral blood was collected from healthy donors and mononuclear cells were isolated using Ficoll density gradient centrifugation.

[0019] The specific procedure is as follows: 50 mL of anticoagulated peripheral blood was slowly added to a 50 mL centrifuge tube containing 15 mL of Ficoll separation buffer, avoiding the formation of bubbles. The cells were centrifuged at 800 g for 20 minutes at room temperature to form distinct layers. The mononuclear cells in the interphase layer were carefully aspirated and transferred to another 50 mL centrifuge tube. The cells were washed twice with DPBS buffer, each time centrifuged at 500 g for 5 minutes. The supernatant was removed to obtain purified mononuclear cells. The mononuclear cells were then pretreated with 30 μg / mL of humanized PD-L1 antibody (atezolizumab) at 37°C for 2 hours to allow the antibody to bind to the cell surface PD-L1 receptor, partially blocking its binding to tumor cell PD-L1 and reducing the inhibitory effect of the tumor microenvironment on NK cells. After pretreatment, the cells were washed twice with DPBS buffer to remove unbound antibody and ensure that subsequent culture steps were not disturbed.

[0020] Flask coating step: Sodium hyaluronate is used in conjunction with antibodies to enhance cell adhesion and activation.

[0021] The specific procedure is as follows: 1.0 mg / mL sodium hyaluronate and 100 μg / mL BCN-labeled anti-human CD16 monoclonal antibody (clone 3G8) are added to a T75 culture flask in a total volume of 20 mL. The culture flask is incubated in a 37°C cell culture incubator for 3 hours to allow the sodium hyaluronate to form a gel network on the flask surface. Simultaneously, the BCN-labeled anti-CD16 antibody adheres to the flask surface through non-covalent interactions. After the incubation period, the culture flask is rinsed three times with PBS buffer to remove unbound sodium hyaluronate and antibody, resulting in a coated flask. Sodium hyaluronate, through its high hydrophilicity and viscoelasticity, provides physical support for the CD16 antibody. Furthermore, its binding to the CD44 receptor on the cell surface further activates NK cells, enhancing cell adhesion and promoting cell activation and proliferation.

[0022] Inoculation and culture steps: The pretreated mononuclear cells are inoculated into the coated culture flask and activated with the addition of a solution to significantly enhance the NK cell expansion efficiency.

[0023] The specific operation is as follows: the washed mononuclear cells were suspended in DPBS buffer and the cell density was adjusted to 1.5×10 6 / mL, seeded in a T75 culture flask; culture medium was added, including 100mL of Lonza X-VIVO 15 serum-free medium as the basal medium; and 6mL of a cytokine cocktail containing the following components in precise proportions: 50ng / mL recombinant human IL-15, 10ng / mL recombinant human IL-21, 50ng / mL recombinant human IL-18, 1μg / mL OK432, 50μg / mL IL-2, 100ng / mL anti-CD2 monoclonal antibody, 100ng / mL anti-NKp46 monoclonal antibody, 5mM N-acetyl-L-cysteine (NAC), and 5μM Galunisertib. The activating solution and basal solution were thoroughly mixed and added to the culture flask, bringing the total volume to 106mL.

[0024] Genetic modification steps: To enhance the specific targeting ability and anti-tumor activity of NK cells, the UniProt P08637 F158V mutant CD16a receptor gene was transduced using a lentiviral vector.

[0025] The specific operation is as follows: On the third day of cell culture, add lentiviral supernatant carrying the humanized CD16a F158V mutant gene at an MOI of 10 to the culture flask; place the culture flask in a 37°C, 5% CO2 incubator for 24 hours to allow the lentivirus to efficiently transduce the cells; after transduction, replace the old culture medium with fresh culture medium (94% basal solution + 6% activation solution) and continue culture.

[0026] Maintenance culture steps: Culture continuously in a 37°C, 5% CO2 incubator for 14 days, and add 50 μg / mL IL-2 every 3 days to maintain cell proliferation and activity.

[0027] The specific procedure is as follows: On day 7 of culture, a DBCO-labeled anti-PD-L1 peptide is added to the culture flask. This peptide is covalently linked to a BCN-labeled anti-CD16 antibody on the cell surface via a copper-free click chemistry reaction (SPAAC). The reaction is incubated at 37°C in the dark for 4 hours. After completion of the reaction, the cells are washed twice with PBS buffer to remove unbound peptide, and fresh culture medium is added for continued culture. This step, through surface engineering, couples the anti-PD-L1 peptide to the cell surface, enhancing the targeted killing ability of NK cells against PD-L1-positive tumors. It also forms a dual targeting mechanism with the genetically modified F158V mutant CD16a receptor, significantly enhancing the anti-tumor effect. During the maintenance culture period, cell counts and viability measurements are performed every 3 days, and cell growth curves are recorded to ensure expansion efficiency. The culture medium is also changed every 3 days to maintain an optimal growth environment for the cells. On the 14th day of culture, cells were collected for flow cytometry analysis to detect the expression levels of CD56, CD16, CD3, and F158V mutant CD16a, and high-purity (CD56⁺CD16⁺CD3⁻, purity ≥90%) NK cells were screened.

[0028] Collection step: NK cells are collected by centrifugation and then subjected to performance testing.

[0029] Example 2

[0030] This example provides a NK cell, which is the same as Example 1 except for the following parameters: Pretreatment step: Anti-PD-L1 antibody (humanized monoclonal antibody, Durvalumab) at a concentration of 35 μg / mL.

[0031] Inoculation and culture steps: The concentration of galunisertib in the culture medium was 6 μM, the concentration of IL-2 was 80 μg / mL, and the concentrations of anti-CD2 monoclonal antibody and anti-NKp46 monoclonal antibody were 500 ng / mL respectively.

[0032] Maintenance culture step: The culture period is 15 days.

[0033] Example 3

[0034] This example provides a NK cell, which is the same as Example 1 except for the following parameters: Pretreatment step: anti-PD-L1 antibody (humanized monoclonal antibody, Avelumab) at a concentration of 25 μg / mL.

[0035] Example 4

[0036] This example provides a NK cell, which is the same as Example 1 except for the following parameters: Inoculation and culture steps: The concentration of Galunisertib in the culture medium was 4 μM.

[0037] Example 5

[0038] This example provides a NK cell, which is the same as Example 1 except for the following parameters: Inoculation and culture steps: The IL-2 concentration in the culture medium was 20 μg / mL.

[0039] Example 6

[0040] This example provides a NK cell, which is the same as Example 1 except for the following parameters: Inoculation and culture steps: The concentration of anti-CD2 monoclonal antibody and anti-NKp46 monoclonal antibody was 300 ng / mL respectively.

[0041] Example 7

[0042] This example provides a NK cell, which is the same as Example 1 except for the following parameters: Pretreatment step: Anti-PD-L1 antibody (humanized monoclonal antibody, Atezolizumab) concentration was 20 μg / mL.

[0043] Example 8

[0044] This example provides a NK cell, which is the same as Example 1 except for the following parameters: Inoculation and culture steps: The concentration of Galunisertib in the culture medium was 3 μM.

[0045] Example 9

[0046] This example provides a NK cell, which is the same as Example 1 except for the following parameters: Inoculation and culture steps: The IL-2 concentration in the culture medium was 10 μg / mL.

[0047] Example 10 This example provides a NK cell, which is the same as Example 1 except for the following parameters: Inoculation and culture steps: The concentration of anti-CD2 monoclonal antibody and anti-NKp46 monoclonal antibody was 50 ng / mL respectively.

[0048] Comparative Example 1 This comparative example provides a NK cell, which is the same as Example 1 except for the following parameters: Omitted pretreatment step: Peripheral blood mononuclear cells (PBMCs) were not pretreated with anti-PD-L1 antibody and were only incubated with DPBS buffer at 37°C for 2 hours.

[0049] Comparative Example 2 This comparative example provides a NK cell, which is the same as Example 1 except for the following parameters: Galunisertib-deficient: The culture medium does not contain Galunisertib and only contains an equal volume of DMSO solvent.

[0050] Comparative Example 3 This comparative example provides a NK cell, which is the same as Example 1 except for the following parameters: Lack of genetic engineering: No lentiviral transduction was performed, and peripheral blood mononuclear cells (PBMCs) were directly cultured.

[0051] Comparative Example 4 This comparative example provides a NK cell, which is the same as Example 1 except for the following parameters: Alternative genetic engineering: lentiviral transduction of wild-type CD16a receptor (UniProt P08637, without F158V mutation) instead of F158V mutant.

[0052] Comparative Example 5 This comparative example provides a NK cell, which is the same as Example 1 except for the following parameters: Absence of antibody coating: The culture vessel was not coated with anti-CD16 antibody and was pretreated with PBS only (simulating the absence of CD16-mediated adhesion and activation).

[0053] Performance testing methods 1. Cell Proliferation and Viability Test Count 1×10⁵ cells and count viable cells using trypan blue staining. Calculate the expansion factor (number of cells at the end of culture / initial seeding number of cells) and viability (ratio of viable cells). Simultaneously, use a CCK-8 kit to assess metabolic activity: seed cells in a 96-well plate (1×10⁴⁴ / well), add CCK-8 solution, incubate at 37°C for 4 hours, and measure absorbance at 450 nm (OD value). Higher OD values indicate greater viability.

[0054] The test results are shown in Table 1 below:

[0055] 2. Cell Purity and Phenotypic Analysis Surface markers were detected by flow cytometry. Cells were stained with anti-CD56-FITC, anti-CD16-PE, and anti-CD3-APC antibodies (incubated in the dark for 30 minutes), washed, and analyzed. The proportion of CD56+CD16+CD3- cells (NK cell purity) was calculated. Furthermore, F158V CD16a expression was detected by staining with anti-CD16a-V450 antibody (specifically recognizing the F158V mutation) and the positive rate was analyzed.

[0056] The test results are shown in Table 2 below:

[0057] 3. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay PD-L1-positive tumor cell lines (e.g., A549 lung cancer cells) were used as target cells. Target cells were labeled with Calcein-AM and co-cultured with effector cells (NK cells) at a 10:1 ratio (target cell:effector cell) for 4 hours. Anti-PD-1 antibody (pembrolizumab, 1 μg / mL) was added to simulate ADCC conditions. After centrifugation, the supernatant was collected and fluorescence intensity was measured (excitation 485 nm, emission 535 nm). Cytotoxicity was calculated as [(experimental group fluorescence - spontaneously released fluorescence) / (maximum released fluorescence - spontaneously released fluorescence)] × 100%. Maximum release was determined by treating target cells with 1% Triton X-100. The test results are shown in Table 3 below.

[0058] 4. Anti-tumor activity test In a Transwell co-culture system, NK cells (1×10⁵) were seeded in the upper layer, and A549 cells (1×10⁵) and Treg cells (1×10⁴, to simulate an immunosuppressive microenvironment) were seeded in the lower layer. The culture medium contained 10 ng / mL TGF-β (to simulate the tumor microenvironment). After 48 hours of culture, cells in the lower layer were harvested, and tumor cell apoptosis was assessed by flow cytometry using Annexin V / PI double staining. The results are shown in Table 3 below.

[0059]

[0060] The above results show that when the anti-PD-L1 antibody concentration is between 25–35 μg / mL, the NK cell purity remains stable at over 90% (Examples 1, 2, and 3), while the purity in Example 7 drops sharply to 80% at concentrations below 25 μg / mL. This indicates that 25 μg / mL is the threshold concentration for effectively blocking PD-L1 / PD-1 immunosuppression: low-concentration pretreatment fails to adequately shield tumor microenvironmental signals, leading to increased T cell contamination during expansion, which in turn impairs expansion fold and metabolic activity. Similarly, galunisertib maintains cytotoxicity >68% at concentrations between 4–6 μM (Examples 1, 2, and 4), while at 3 μM in Example 8, the purity drops to 50%. Insufficient inhibition of TGF-β signaling exacerbates tumor microenvironment-induced NK cell exhaustion and significantly reduces ADCC activity, indicating that 4 μM is the critical threshold for effective inhibition of this pathway.

[0061] In Examples 1-2, lentiviral transduction resulted in F158V expression reaching 88–90%. Combined with the sustained activation signal provided by anti-CD16 antibody coating, ADCC activity increased to 75–78%. While Comparative Example 3 retained basal CD16 expression (5% background on flow cytometry), cytotoxicity plummeted to 30%. Even with expression of the mutant receptor in Comparative Example 5, metabolic activity (OD450 1.10) remained near basal levels due to the lack of adhesion and activation signals. This demonstrates that cross-linking of the mutant receptor with CD16 is required for maximal ADCC efficacy. Furthermore, the anti-PD-L1 peptide conjugated to the cell surface in Example 1 (maintained during the culture step) immediately neutralized tumor PD-L1, resulting in a 70% apoptosis rate, double that of Comparative Example 1.

[0062] Comparative Example 1 (no PD-L1 pretreatment) exhibited only a 100-fold expansion and a 35% apoptosis rate due to the lack of relief of initial immunosuppression. Under TGF-β stress, cell viability in Comparative Example 2 (no galunisertib) dropped to 78%, demonstrating that TGF-β inhibition is essential for maintaining cell viability. Comparative Example 4 (wild-type CD16a), despite transduction with the wild-type receptor, exhibited far lower ADCC activity (50%) than the F158V mutant group (75%). Comparative Example 3 (no genetic modification) exhibited a "false positive" phenomenon: its NK purity (85%) approached that of the optimal group, but F158V expression was only 5%, and its cytotoxicity (30%) was inconsistent with the purity.

[0063] Application Example: Anti-tumor Effect of NK Cells in Tumor-Bearing Mouse Model Animal Model: 6-8 week-old BALB / c nude mice (n=10 / group) were subcutaneously inoculated with human PD-L1-positive A549 lung cancer cells (5×10⁶ / mouse). When tumor volume reached 100-150 mm³, mice were divided into two groups: a control group (PBS) and an experimental group (NK cells prepared in Example 1 + anti-PD-1 antibody).

[0064] Dosing regimen: Mice in the experimental group received tail vein injections of NK cells (5×10⁶ cells / mouse) and pembrolizumab (10 μg / mouse) at a 1:2 ratio (10⁶ cells:μg antibody) once weekly for three doses. After cell recovery, the cells were preactivated for 2 hours with a tumor microenvironment-specific activation solution (containing 100 ng / mL IL-15, 50 ng / mL IL-23, 20 ng / mL IL-18, and 100 μg / mL OK432).

[0065] Monitoring indicators: Tumor volume: The volume was calculated by measuring with a vernier caliper every 3 days (length × width² × 0.5).

[0066] Survival rate: Record the survival status within 60 days.

[0067] Tissue analysis: Mice were sacrificed, and tumors and spleens were removed. NK cell infiltration (anti-CD56 antibody), PD-L1 expression, and apoptosis (TUNEL staining) were detected by flow cytometry.

[0068] result: Tumor volume: The tumor growth inhibition rate in the experimental group was 85% (vs control group, P<0.01), and the volume on day 21 was 120±15 mm³ (450±50 mm³ in the control group).

[0069] Survival rate: The 60-day survival rate of the experimental group was 90%, while that of the control group was 0% (all died).

[0070] Tissue analysis: NK cell infiltration in the experimental group tumors increased (CD56+ cell ratio 25±5%), PD-L1 expression was downregulated, and the proportion of apoptotic cells was 40±5%.

[0071] Conclusion: The NK cells prepared in Example 1 combined with anti-PD-1 antibodies significantly inhibited tumor growth and prolonged survival, confirming its application value in the preparation of anti-tumor drugs.

[0072] The above description is merely a preferred embodiment of the present disclosure and an illustration of the technical principles employed. Those skilled in the art should understand that the scope of the invention herein is not limited to technical solutions formed by specific combinations of the aforementioned technical features. It also encompasses other technical solutions formed by any combination of the aforementioned technical features or their equivalents, without departing from the inventive concept. For example, a technical solution formed by replacing the aforementioned features with (but not limited to) technical features with similar functions disclosed in this disclosure.

Claims

1. A NK cell, characterized in that Prepared by the following method: Step a: Pre-treat human peripheral blood mononuclear cells with anti-PD-L1 antibody at a concentration of 20–35 μg / mL; Step b: Incubate cells in an anti-CD16 antibody-coated container with medium containing galunisertib at a concentration of 3–6 μM. Step c: Genetically engineer the CD16a receptor expressing the F158V mutant of UniProt P08637.

2. The NK cell according to claim 1, wherein: The anti-PD-L1 antibody in step a is a humanized monoclonal antibody.

3. The NK cell according to claim 1, wherein: The concentration of galunisertib in step b was 4–6 μM.

4. The NK cell according to claim 1, wherein: In step c, the genetic engineering modification is achieved by lentiviral vector transduction.

5. The NK cell according to claim 1, wherein: The culture medium in step b contains IL-2 at a concentration of 20-80 μg / mL.

6. The NK cell according to claim 1, wherein: The anti-PD-1 antibody is a PD-1 / PD-L1 pathway specific blocking antibody.

7. The NK cell according to claim 1, wherein: The culture medium in step b contains anti-CD2 monoclonal antibody at a concentration of 100-500 ng / mL and anti-NKp46 monoclonal antibody at a concentration of 100-500 ng / mL.

8. The NK cell according to claim 1, wherein: The culture period is 13–15 days.

9. Use of the NK cells according to any one of claims 1 to 8 in the preparation of drugs for treating tumors.

10. Use of the NK cells according to claim 9 in preparing a drug for treating tumors, wherein: The drug includes NK cells and anti-PD-1 antibodies.

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